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A kind of car-nk cell and its preparation method and application

A cell and cell culture technology, applied in the field of CAR-NK cells and their preparation, can solve the problems of instability and transduction efficiency less than 10%, and achieve high stability, best industrial application and clinical application value, and transfection efficiency stable effect

Active Publication Date: 2020-07-03
深圳市沃英达生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transduction efficiency of liposome transfection and electroporation transfection is less than 10%, and the efficiency of lentiviral transfection is 12% to 73%, which is not stable

Method used

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  • A kind of car-nk cell and its preparation method and application
  • A kind of car-nk cell and its preparation method and application
  • A kind of car-nk cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A preparation method of CAR-NK cells, the preparation method comprising the following steps;

[0053] S1: Artificially synthesized CAR sequence CD19scFv--CD28--4-1BB--FcεRIγ, which is shown in the base sequence of CD19scFv---CD28---4-1BB---FcεRIγ in the sequence table; The intracellular signal segment is used as the intracellular signal segment of CAR, CD28 is used as the transmembrane region, and 4-1BB is used as a co-stimulatory factor to construct a CAR targeting CD19;

[0054] S2: Integrate the CAR sequence CD19scFv--CD28--4-1BB--FcεRIγ synthesized in step S1 into the lentiviral target vector plasmid pWPXL-GFP to obtain the target plasmid, denoted as pWPXL-FcεRIγ;

[0055] S3: (1) Add 450 μl ddH2O to a 1.5ml sterile EP tube, add 10 μg of the target plasmid pWPXL-FcεRIγ, 6.5 μg of the packaging plasmid pMD2.G, and 3.5 μg of the envelope plasmid PSPAX2; (2) Slowly add 50 μl of CaCl2 and mix gently Then, add the mixture containing CaCl2 and plasmid to 500 μl 2x HBS, m...

Embodiment 2

[0062] 1. Using the intracellular signal segment of the NK cell activating receptor FcεRIγ as the intracellular signal segment of the CAR, CD28 as the transmembrane region, and 4-1BB as the co-stimulatory factor, a CAR targeting CD19 was constructed, and the structure was designed as CD19scFv- --CD28---4-1BB---FcεRIγ, and construct a CAR with CD3ζ as the intracellular signal segment: CD19scFv---CD28---4-1BB---CD3ζ as a control. After artificially synthesizing CD19scFv---CD28---4-1BB---FcεRIγ and CD19scFv---CD28---4-1BB---CD3ζ, CD19scFv---CD28---4-1BB---CD3ζ The sequence of CD19scFv --- CD28 --- 4-1BB --- CD3ζ base sequence shown in the sequence list, these two sequences were integrated into the lentiviral target vector plasmid pWPXL-GFP, respectively named as the target plasmid pWPXL -FcεRIγ and the target plasmid pWPXL-CD3ζ.

[0063] 2. Take 5x10 293T cells in the logarithmic growth phase 6 Each was inoculated in a 100mm cell culture dish. After 24 hours, when the cell conf...

Embodiment 3

[0081] Modify the polypeptide sequence in Example 1 or Example 2 to Ac-NIFDITNILIYIK-NH 2 , without changing any other steps, reagents, parameters, etc., the transfection rate is only about 20-40%.

[0082] In the present invention, the amino acid sequence expressed by the base sequence of CD19scFv---CD28---4-1BB---FcεRIγ is as expressed by "CD19scFv---CD28---4-1BB---FcεRIγ" in the sequence table The amino acid sequence of CD19scFv --- CD28 --- 4-1BB --- CD3ζ base sequence is shown in "CD19scFv --- CD28 --- 4-1BB --- CD3ζ The expressed amino acid sequence" is shown.

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Abstract

The invention relates to a CAR-NK cell, and a preparation method and an application thereof. The preparation method comprises the following steps: S1, synthesizing a CAR sequence; S2, integrating theCAR sequence into a lentiviral target vector plasmid to obtain a target plasmid; S3, mixing the target plasmid with a 293T cell, performing culturing, collecting the obtained virus liquid, and concentrating the virus liquid to obtain the virus concentrate of the target plasmid; S4, collecting autologous plasma and peripheral blood mononuclear cells (PBMC), adjusting the cell density with an activation culture medium, performing cell culturing, transferring obtained cells into a new cell culturing bag, continuing culturing, adding a proliferation culturing medium, and obtaining the NK cell after the culturing is finished; and S5, adding the virus concentrate of the target plasmid to the NK cell, and performing incubation to obtain the CAR-NK cell. The CAR-NK cell has very strong factor secretion ability and tumor inhibition ability, and the transfection method for preparing the CAR-NK cell is efficient and stable.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CAR-NK cell and its preparation method and application. Background technique [0002] Natural killer cells (Nature killer cells, NK cells) are an important natural immune system lymphocytes in the body. Its phenotype is generally CD3-CD16+CD56+, mainly derived from bone marrow CD34+ lymphocytes. NK cells are not limited by MHC and can recognize and kill tumor cells without antigen sensitization. At the same time, NK cells can secrete a variety of cytokines to regulate the acquired immune response, and are a bridge connecting the innate immune response and the acquired immune response, so NK cells play an important role in the immune response against tumors and viral infections, known as The first line of defense for human health. [0003] The recognition of tumor cells by NK cells mainly depends on the receptors on the cell surface and is not restricted by MHC. After recognizin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/2803C07K2319/02C07K2319/03C07K2319/33C12N5/0646C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 王旭李陶林词雄林洁璇朱刚
Owner 深圳市沃英达生命科学有限公司
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